The present invention concerns prodrugs whose aromatic oxidation, particularly their enzymatic aromatic hydroxylation, results in their activation by the release of a drug moiety. It particularly concerns anti-tumour prodrugs and those which are specifically activated by the hydroxylation activity of the P-450 enzyme CYP1B1.
Many conventional cytotoxic drugs are known (for example colchicine, esperimycin, taxol, daunomycin and staurosporin) which can be used for chemotherapeutic purposes. However, they typically suffer from the problem that they are generally cytotoxic and therefore may affect cells other than those which it is wished to target. This can be alleviated somewhat by using targeted drug delivery systems, for example direct injection to a site of tumorous tissue, or by e.g. binding the cytotoxic agent to antibody which specifically recognises an antigen displayed by cancerous cells. Alternatively, electromagnetic radiation may be used to cause chemical changes in an agent at a desired site in the body such that it becomes cytotoxic. However, all of these techniques have, to a greater or lesser extent, certain limitations and disadvantages.
It has been reported (Murray, G. I. et al., Jul. 15, 1997. Cancer Research, 57: 3026-3031) that the enzyme CYP1B1, a member of the cytochrome P450 family of xenobiotic metabolizing enzymes, is expressed at a high frequency in a range of human cancers including cancers of the breast, colon, lung, oesophazus, skin, lymph node, brain and testis, and that it is not detectable in normal tissues. This led to the conclusion (p. 3030, final sentence) that xe2x80x9c. . . the expression of CYP1B1 in tumour cells provides a molecular target for the development of new anticancer drugs that could be selectively activated by the presence of CYP1B1 in tumour cellsxe2x80x9d. It was also reported (p.3030, column 1 lines 15-17) that CYP1B1 is capable of 4-hydroxylation of estradiol. No specific anticancer drugs were suggested.
The present inventors have now succeeded in creating a range of prodrugs having a xe2x80x9ccarrierxe2x80x9d framework with a drug moiety conjugated to it (the prodrug other than the drug moiety is referred to below as xe2x80x9cthe rest of the prodrugxe2x80x9d) which have little or no cytotoxic effect when in their normal state, but whose aromatic oxidation e.g. hydroxylation (for example by CYP1B1) results in the release of the drug moiety. With CYP1B1 as a hydroxylating enzyme, this provides for a self-targeting drugs delivery system in which a non-cytotoxic (or at least negligibly cytotoxic) compound can be administered to a patient, for example in a systemic manner, the compound then being hydroxylated at the site of tumour cells (intratumoural hydroxylation) to release the drug which acts to kill or otherwise affect the tumour cells. The fact that CYP1B1 is not expressed by normal cells means that the hydroxylation of the prodrug only occurs at the site of tumour cells and therefore only tumour cells are affected, thus providing a self-targeting drug delivery system.
The prodrugs of the present invention have the distinct advantage of being useful in the treatment of tumours at any site in the body, meaning that even tumours which have undergone metastasis (which are not normally susceptible to site-specific therapies) may be treated, as well of course as primary and secondary tumours.
The prodrugs may be designed to be activated by other oxidising agents, for example other enzymes (e.g. other members of the cytochrome P-450 family of enzymes) which cause hydroxylation of the prodrug. For example, a prodrug activated by hydroxylation by the human CYP1A1 isoform would be useful for the treatment of stomach cancer since this isoform is over expressed in this type of cancer (Murray et al., 1998, Br. J. Cancer, 77: 1040). Furthermore, if the prodrug is specifically activated by a fungal P-450 enzyme isoform then it has utility as a selective antifungal agent, and similarly a prodrug specifically activated by a bacterial P-450 enzyme isoform would have utility as a selective antibiotic agent.
CYP1B1 has not yet been fully characterised, and it is therefore possible that tumour-specific isoforms of it may exist which possess the same catalytic properties. The prodrugs of the present invention may, of course, be used with such enzymes.
In the case of cytochrome P-450 activated prodrugs, the therapeutic strategy achieved using them is referred to as SPEAR (Specific P-450 Enzyme Activated drug Release).
According to the present invention there is provided a prodrug comprising a drug moiety bound to a carrier framework, the prodrug being activated by aromatic oxidation of the carrier framework to release the drug moiety.
The prodrug may be activated by aromatic hydroxylation. It may be activated by enzymatic aromatic hydroxylation.
Other enzymatically-activated prodrugs are known, for example those which release a drug moiety as the result of cleavage by a peptidase enzyme. However, nowhere has it been previously suggested that a prodrug could be activated to release a drug moiety by enzymatic hydroxylation.
A prodrug according to the present invention may have the formula (Z): 
wherein:
X=H, OH, OMe or N(CH3)2; and
n=0-6;
and:
R1=H, C1-4 lower alkyl, or together with R2 forms part of a cycloalkyl group which may be further substituted to form part of a polycyclic cycloalkyl group, or with R2 forms part of a steroidal carbon framework;
R2=H, OMe, C1-4 lower alkyl, or together with R1 and/or R3 forms part of a cycloalkyl, polycyclic cycloalkyl or steroidal carbon framework, or forms part of a polycyclic aromatic group by linkage to R4;
R3=H, OMe, C1-4 lower alkyl or together with R2 forms part of a cycloalkyl, polycyclic cycloalkyl or steroidal carbon framework; and
R4=H or is fused directly to the aromatic position designated by R2 and either:
the drug moiety is derived from a drug having a free amino, hydroxyl or thiol group and which links it to the rest of the prodrug, such that A represents NH, NR (R=C1-4 lower alkyl), O or S; or
the drug moiety is derived from a drug having a carboxylate group, an ester linkage joining it to the rest of the prodrug and A being absent.
Enzymatic hydroxylation of the prodrugs of formula (Z) results in the transfer of electrons from the site of hydroxylation (for example the aromatic 4 positionxe2x80x94see FIG. 1) to the drug moiety, resulting in its release.
The prodrug may, for example, be an anti-tumour prodrug. The drug moiety may be cytotoxic or cytostatic, although of course it may be a moiety which has any other desired effect. Examples of classes of drug moiety include antimitotic agents, alkylating agents, antifolates, antimetabolites. DNA-damaging agents and enzyme inhibitors. Specific examples of possible cytotoxic drug moieties include 5-fluorouracil, colchicine, esperimycin, taxol, daunomycin, staurosporin, and nitrogen mustard. Alternatively, the drug moiety could be e.g. a fluorescent organic molecule which would be released in an intratumoural manner, aiding tumour detection by correlating specific cell fluorescence with the presence of the drug moiety and thus of the oxidising agent (e.g. CYP1B1) which caused its release.
Thus the term xe2x80x9cdrugxe2x80x9d also extends to moieties which may be used for diagnostic purposes.
A possible nitrogen mustard is, for example, a para-hydroxy aniline mustard that is linked through the para-hydroxy group to the rest of the prodrug. In the case of nitrogen mustard prodrugs, the mustard function is itself activated only when the drug moiety is released from the prodrug. Another example of a nitrogen mustard which can be incorporated into a SPEAR prodrug is Nor-mustine, which can be linked directly through the mustard nitrogen atom. In this case the carbamate linked nor-mustine prodrug has very low toxicity, but upon enzymatic hydroxylation of the prodrug the potent cytotoxic agent Nor-mustine is released.
The olefin linkage 
may have a cis- or trans-geometry. It may be acyclic or cyclic. It may form part of an aromatic or polycyclic aromatic system.
The prodrug may be activated by CYP1B1. Thus a prodrug which releases a cytotoxic drug moiety upon hydroxylation by CYP1B1 may be used as a self-targeting anti-tumour drug, being activated at the site of a tumour by CYP1B1 and having no (or negligible) cytotoxicity in the rest of the body.
The linkage to the drug moiety from the carrier framework may be from a hydroxyalkyl group in the prodrug via a carbamate, carbonate or thiocarbonate linker to an amino, hydroxy or thiol group in the drug moiety.
Using the strategy and prodrugs of the present invention, it is possible to link any desired drug moiety through a free amino, hydroxy or thiol group. The provision of a linker group comprising a carbamate, carbonate or thiocarbonate linker joining the drug moiety to the rest of the prodrug results in the release of carbon dioxide upon release of the drug moiety, making the reaction irreversible. Thus the hydroxylation (or other aromatic oxidation) of the prodrug may cause the release of the drug moiety and carbon dioxide.
A prodrug may have a steroid carbon carrier framework. For example, it may be derived from estradiol.
An example of a prodrug according to the present invention is the prodrug having the formula I, shown in FIG. 1. It is an estradiol derivative and incorporates the drug moiety at the steroid 6-position. In this position, the 3-hydroxy group of estradiol does not provide the requisite electron release, but upon 4-hydroxylation the electron release from the 4-hydroxy group triggers electron transfer within the prodrug, resulting in the release of the drug moiety.
A prodrug according to the present invention may, for example, have the formula of any one of formulae (I)-(IX): 
wherein xe2x80x94OR=xe2x80x94Ome or xe2x80x94OH
The prodrug may have the formula of any one of formulae (X)-(XIII): 
where R=H (Formula XIIa) or R=Me (Formula IIb)
Formula (X) is a colchicine-estradiol prodrug; (XI) is a combretastatin-estradiol prodrug; (XII) is a mustard-estradiol prodrug; (XIII) is a fluorophore-estradiol conjugate. The prodrug may have a polycyclic aromatic carrier framework. For example the prodrug may be based on the naphthyl or phenanthryl structures. Examples of naphthyl based prodrugs and phenanthryl based prodrugs are given by the formulae (XIV) to (XVII) below. The compound (XIV) is a naphthyl/colchicine prodrug, (XV) is a naphthyl/mustard prodrug, (XVI) is a naphthyl/5-fluorouracil prodrug, and (XVII) is a phenanthryl/mustard prodrug. The synthetic route used for the synthesis of the naphthyl based prodrugs is outlined in the scheme shown in FIG. 3. 
The prodrug may be based on a substituted benzyl carrier framework. For example the prodrug may be based on various methoxy substituted benzyl groups, and these are exemplified by the benzyl/mustard compounds having formulae (XVIII) to (XXII). Compound (XVIII) is a 3-methoxybenzyl/mustard prodrug, (XIX) is a 3,5-dimethoxybenzyl/mustard prodrug, (XX) is a benzyl/mustard prodrug, (XXI) is a 2-methoxybenzyl/mustard prodrug, and (XXII) is a 2,5-dimethoxybenzvl/mustard prodrug. These compounds have the general formula (Y): 
Specific molecules having formulae (XVIII)-(XXII) have groups R2, R3 and X as detailed below:
The variety of drugs and fluorophores which may be linked to the benzyl carrier framework is further exemplified by the benzyl based prodrugs given in formulae (XXIII) to (XXVIII). Compound (XXIII) is a 3-methoxybenzyl/5-fluorouracil prodrug, (XXIV) is a 3-methoxybenzyl/colchicine prodrug, (XXV) is a 3-methoxybenzyl/calchone prodrug derived from the cytotoxic calchone (E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-1-en-3-one, (XXVI) is a 3-methoxybenzyl/combretastatin prodrug, (XXVII) is a 3-methoxybenzyl/resorufin fluorophore conjugate, and (XXVIII) is a 3-methoxybenzyl/7-amino-4-methylcoumarin fluorophore conjugate. 
The prodrug may be based on the cinnamyl carrier framework, and this is exemplified by the cinnamyl based prodrugs given by formulae (XXX) to (XXXII). Compound (XXX) is a 3-methoxycinnamyl/mustard prodrug, (XXXI) is a cinnamyl/resorufin fluorophore conjugate, and (XXXII) is a cinnamyl/7-amino-4-methylcoumarin fluorophore conjugate. 
Also provided according to the present invention is a prodrug according to the present invention for use in a method of treatment or diagnosis of the human or animal body, particularly the treatment or diagnosis of tumours.
Also provided according to the present invention is the use of a prodrug according to the present invention in the manufacture of a medicament, e.g. for the treatment of tumours.
Also provided according to the present invention is a method of manufacture of a medicament, comprising the use of a prodrug according to the present invention.
Also provided according to the present invention is a method of treatment of a patient, comprising administering to the patient a prodrug according to the present invention. The prodrug may be administered to treat a medical condition e.g. an illness.
Methods of manufacture of medicaments are well known. For example a medicament may additionally comprise a pharmaceutically acceptable carrier diluent or excipient (Remington""s Pharmaceutical Sciences and US Pharmacopeia, 1984, Mack Publishing Company, Easton, Pa. USA).
The exact dose (i.e. a pharmaceutically acceptable dose) of prodrug to be administered to a patient may be readily determined by one skilled in the art, for example by the use of simple dose-response experiments.
Since prodrugs of the present invention may be specific to e.g. tumour cells, they may not only be used to treat tumours, but may also be used to determine whether or not a patient (or a sample taken from a patient) has tumour cells. For example, tumour cells may be detected by using a SPEAR prodrug that is a fluorophore conjugate which releases a fluorescent compound upon enzymatic hydroxylation. An example of this type of fluorophore conjugate is given by compound (XIII). Cell numbers in a sample may be assayed, as may the presence and quantity of the oxidised e.g. hydroxylated prodrug, thus providing for the diagnosis of the presence of tumour cells. Another way in which a SPEAR prodrug may be used for diagnosis is by using a carbon-13 isotopically labelled carbonyl linkage. Here the carbon dioxide liberated following aromatic hydroxylation of the prodrug will contain the carbon-13 isotope and can thus be measured by a carbon-13 detection technique such as mass spectrometry. The carbon-13 labelled carbon dioxide would be detectable in the exhaled breath of a patient administered with the prodrug, and therefore this technique provides a means for a diagnostic breath test for cancer. Thus in any diagnostic test, the drug moiety may be anything which, upon aromatic oxidation of the prodrug, results in the release of a detectable product. Thus in a diagnostic test, a detection step may be for any product of aromatic oxidation of the prodrug, for example the drug moiety, carrier framework or other product of aromatic oxidation.
Thus the present invention also provides a method of detection of aromatic oxidation, comprising the steps of:
i) contacting a sample with a prodrug according to any one of claims 1-26;
ii) detecting any product of aromatic oxidation of the prodrug; and
iii) correlating detection of the product of aromatic oxidation of the prodrug with aromatic oxidation activity.
The aromatic oxidation activity may be enzymatic, for example CYP1B1 aromatic oxidation activity.
The method may be a method of detection of tumour cells.
The method may be a method of diagnosis of the human or animal body.